The first portion of the competing continuation project will examine the effects of acute and chronic ethanol and barbiturate administration on fast- and slow phase, voltage-dependent 45Ca++ entry into and endogenous neurotransmitter release from brain synaptosomes. Recent evidence suggests that the fast-component, which terminates within a few seconds following depolarization, correlates closely with physiologically operative processes involved in calcium-dependent neurotransmitter release in brain. The proposed studies will examine the acute and chronic effects of ethanol and barbiturates on 45Ca++ entry and neurotransmitter release in seven brain regions: cerebral cortex, brain stem, cerebellum, midbrain, hypothalamus, striatum and hippocampus. Endogenous neurotransmitter release studies will measure fast- and slow phase norepinephrine, dopamine and acetylcholine release. Assay procedures have been developed for this project which allow for the simultaneous measurement of calcium entry and endogenous neurotransmitter release in the same synaptosomal samples. The objective of the above investigations will be to determine the changes in voltage-dependent calcium entry and neurotransmitter release in the proposed seven brain regions which correlate with intoxication, tolerance development and/or physical dependence production. Synaptosomes from tolerant ethanol and barbiturate animals will also be analyzed for cross-tolerance to ethanol and barbiturate induced inhibition of calcium uptake and neurotransmitter release. The second phase of the proposed project will correlate changes in free cytosolic calcium concentrations (using quin-2) in the above brain regions with intoxication, tolerance development and physical dependence production. Parallel studies will examine ethanol and/or barbiturate induced changes in ATP-dependent and respiration-linked calcium transport by synaptic plasma membranes, microsomes and mitochondria. The overall objective of our proposed studies will be to determine cellular mechanisms involved in the production of sedation and the development of tolerance and physical dependence.